Riboflavin, also referred to as vitamin B.sub.2, is the precursor of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), essential cofactors for a number of mainstream metabolic enzymes that mediate hydride, oxygen, and electron transfer reactions. Riboflavin-dependent enzymes include succinate dehydrogenase, NADH dehydrogenase, ferredoxin-NADP.sup.+ oxidoreductase, acyl-CoA dehydrogenase, and the pyruvate dehydrogenase complex. Consequently, fatty acid oxidation, the TCA cycle, mitochondrial electron-transport, photosynthesis, and numerous other cellular processes are critically dependent on either FMN or FAD as prosthetic groups. Other notable flavoproteins include glutathione reductase, glycolate oxidase, P450 oxido-reductase, squalene epoxidase, dihydroorotate dehydrogenase, and a-glycerophosphate dehydrogenase. Genetic disruption of riboflavin biosynthesis in Escherichia coli (Richter et al., J. Bacteriol. 174:4050-4056 (1992)) and Saccharomyces cerevisiae (Santos et al., J. Biol. Chem. 270:437444 (1995)) results in a lethal phenotype that is only overcome by riboflavin supplementation. This is not surprising, considering the ensemble of deleterious pleiotropic effects that would occur with riboflavin deprivation.
Riboflavin is synthesized by plants and numerous microorganisms, including bacteria and fungi (Bacher, A., Chemistry and Biochemistry of Flavoproteins (Muller, F., ed.) vol. 1, pp. 215-259, Chemical Rubber Co., Boca Raton, Fla. (1991)). Since birds, mammals, and other higher organisms are unable to synthesize the vitamin and, instead, rely on its dietary ingestion to meet their metabolic needs, the enzymes that are responsible for riboflavin biosynthesis are favorable targets for future antibiotics, fungicides, and herbicides as they should have no adverse affects on such nontarget organisms. Moreover, it is possible that the distantly-related plant and microbial enzymes have distinct characteristics that could be exploited in the development of potent organism-specific inhibitors. Thus, a detailed understanding of the structure, mechanism, kinetics, and substrate-binding properties of the riboflavin biosynthetic enzyme(s), from plants for example, would serve as a starting point for the rational design of chemical compounds that might be useful as herbicides. Having the authentic fungal or plant protein(s) in hand would also provide a valuable tool for the in vitro screening of chemical libraries in search of riboflavin biosynthesis inhibitors.
Fungal and bacterial riboflavin biosynthesis has been intensively studied for more than four decades (For recent reviews, see Bacher, A., Chemistry and Biochemistry of flavoproteins (Muller, F., ed.) vol. 1, pp. 215-259 and 293-316, Chemical Rubber Co., Boca Raton, Fla. (1990)). The synthetic pathway consists of seven distinct enzyme catalyzed reactions, with guanosine 5'-triphosphate (GTP) being the foremost precursor. ##STR1##
While the second and third steps of riboflavin biosynthesis occur in opposite order in bacteria and fungi, the remaining pathway intermediates are identical in both microorganisms. Lumazine synthase (LS), the penultimate enzyme of riboflavin biosynthesis, catalyzes the condensation of 3,4-dihydroxy-2-butanone 4-phosphate (DHBP) with 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine (RAADP) to yield 1 mol each of orthophosphate and 6,7-dimethyl-8-(1'-D-ribityl)-lumazine (DMRL), the immediate precursor of riboflavin. The terminal step of riboflavin biosynthesis is mediated by riboflavin synthase (RS). This enzyme catalyzes the dismutation of two molecules of MRL to yield 1 mol of riboflavin and RAADP.
Ribulose 5-phosphate serves as substrate for the formation of DHBP catalyzed by the enzyme 3,4-dihydroxy-2-butanone 4-phosphate synthase (DS). The complex enzyme reaction involving DS entails the elimination of C-4 from Ribulose 5-phosphate as formate via an intramolecular rearrangement as well as the conversion of the position 1 hydroxymethyl group to a methyl group. The catalytic process probably involves a sequence of tautomerization reactions. It is remarkable that such a complex reaction can be performed by a single and relatively small protein !
DS-encoding genes have been cloned from numerous organisms, including Escherichia coli (GenBank accession number X66720; Richter et al., J. Bacteriol. 174:40504056 (1992)), Vibrio harvey (GenBank accession number M27139; Swartztan et al., J. Biol. Chem. 265:3513-3517 (1989)), Photobacterium phosphoreum (GenBank accession number L11391; Lee et al., J. Bacteriol. 176:2100-2104 (1994)), Bacillus substilis (GenBank accession number X51510; Kil et al., Mol. Gen. Genet. 233:483-486 (1992)), Bacillus amyloliquefaciens (GenBank accession number X95955; Gusarov et al., Mol. Biol. 31:370-376 (1997)), Actinobacillus pleuropneumoniae (GenBank accession number U27202; Fuller et al., J. Bacteriol. 177:7265-7270 (1995)), Saccharomyces cerevisiae (GenBank accession number Z21619; Revuelta, J. L., direct submission; WO 9411515) and Ashbya gossypii (DGene accession number 95N-T03516; DE 4420785). While the various DS homologs all share certain structural features in common, their overall homology at the primary amino acid level is rather poor. For example, as determined with the Genetics Computer Group Gap program (Wisconsin Package Version 9.0, Genetics Computer Group (GCG), Madison, Wis. using their standard default values for "gap creation penalty" of 12 and "gap extension penalty" of 4), the Escherichia coli is only 61%, 25%, 16%, 27%, 33%, 45% and 43% identical to the homologous proteins of Vibrio harveyi, Photobacterium phosphoreum, Bacillus substilis, Bacillus amyloliquefaciens, Actinobacillus pleuropneumoniae, Saccharomyces cerevisiae and Ashbya gossypii, respectively. In addition, pairwise comparisons of these eight proteins reveal that the two most similar homologs share only 61% identity. The only known isolated fungal DS genes are that of Ashbya gossypii and Saccharomyces cerevisiae.
From the foregoing discussion, it is apparent that too little is known about fungal DS genes/proteins and their relationship to known microbial homologs to allow isolation of DS-encoding genes from any fungal or plant species using most classical approaches. The latter include hybridization probing of cDNA libraries with homologous or heterologous genes, PCR-amplification of the gene of interest using oligionucleotide primers corresponding to conserved amino acid sequence motifs, and/or immunological detection of expressed cDNA inserts in microbial hosts. Unfortunately, these techniques would not be expected to be very useful for the isolation of fungal or plant DS genes, since they all heavily rely on the presence of significant structural similarity (i.e., DNA or amino acid sequence) with known proteins and genes that have the same function. Given the observation that DS proteins are so poorly conserved, even amongst microorganisms, it is highly unlikely that the known microbial homologs would share significant structural similarities with their counterparts in higher plants.
An alternative approach that has been used to clone biosynthetic genes in other metabolic pathways from higher eucaryotes is through complementation of microbial mutants that are deficient in the enzyme activity of interest. Since this strategy relies only on the functional similarity between the disrupted host protein and the target gene of interest, it is ideally suited for cloning structurally dissimilar proteins that catalyze the same reaction. For functional complementation, a cDNA library is constructed in a vector that can direct the expression of the cDNA in the microbial host. The plasmid library is then introduced into the mutant microbe, and colonies are selected that are no longer phenotypically mutant. Indeed, the arabidopsis GTP cyclohydrolase II (Kobayashi et al, Gene 160:303-304 (1995)), LS (Garcia-Ramirez et al., J. Biol. Chem. 270:23801-23807 (1995)) and RS (Santos et al., J. Biol. Chem. 270:437444 (1995)) of yeast, were all cloned through functional complementation of microbial riboflavin auxotrophs. This strategy has also worked for isolating genes from higher eucaryotes that are involved in other metabolic pathways, including lysine biosynthesis (Frisch et al., Mol. Gen. Genet. 228:287-293 (1991)), purine biosynthesis (Aimi et al., J. Biol. Chem. 265:9011-9014 (1990)), and tryptophan biosynthesis (Niyogi et al., Plant Cell 5:1011-1027 (1993)), and has also been successfully employed in the isolation of various plant genes including glutamine synthetase (Snustad et al., Genetics 120:1111-1124 (1988)), pyrroline-5-carboxylate reductase (Delauney et al., Mol. Genet. 221:299-305 (1990)), dihydrodipicolinate synthase (Frisch et al., Mol. Gen. Genet. 228:287-293 (1991)), 3-isopropylmalate dehydrogenase (Ellerstrom et al., Plant Mol. Biol. 18:557-566 (1992)), and dihydroorotate dehydrogenase (Minet et al., Plant J. 2:417422 (1992)).
Despite the obvious attractive features of cloning by functional complementation, there are several reasons why this approach might not work when applied to a fungal DS gene. First, the fungal cDNA sequence might not be expressed at adequate levels in the mutant microbe for a variety of reasons, including differences in preferred codon usage. Second, the cloned DS gene might not produce a functional polypeptide, if for instance, enzyme activity requires a post-translational modification, such as acetylation, glycosylation, or phosphorylation that is not carried out by the microbial host. Third, the heterologous fungal protein might be lethal to the host, thus rendering its expression impossible. Fourth, the fungal protein might fail to achieve its native conformation in the foreign microbial environment, due to folding problems, inclusion body formation, or various other reasons. If any of these events were to occur, cloning the DS gene by functional complementation would not be possible.